During the first half of the 1990's many research groups worked departing from the hypothesis that autologous tumor cells (taken from the same human being) could be reformed to be potent antigen-presenting cells (APCs) through genetic modification. In vivo studies in mice were reported in which tumor cells, transfected ex vivo with genes coding for the co-stimulatory molecule B7, used as cancer vaccine by injecting these cells into the recipient had some success. The hypothesis was that the tumor cell not only would express signal 1 (MHC class I+tumor peptide) for CD8+ tumor specific T cells but also signal 2, B7, which theoretically could lead to an efficient activation of these tumor reactive CD 8+ CTL (cytotoxic T lymphocyte).
During a critical survey of the underlying immunological mechanisms responsible for the often very positive effects of such vaccination protocol, it was clearly shown that professional APCs of the host, rather than the vaccinating tumor cells themselves, were responsible for CTL priming. Most likely tumor cells expressing B7 were efficiently killed by natural killer (NK)-cells. This NK-cell mediated immune response has further been shown to induce a local recruitment of host antigen-presenting cells (APCs), including dendritic cells (DCs), whereby these cells take up whole cellular proteins released into the tumor's microenvironment and present them indirectly to CTL, so called cross-priming. This indirect pathway of antigen presentation also explain why vaccination with tumor cells transfected with the gene coding for granulocyte-macrophage colony stimulating factor (GM-CSF) induce a reasonably good anti-tumor response in rodent models since local GM-CSF production have been shown to induce a local production of macrophage inflammatory protein (MIP)-1 alpha which is a strong chemoattractant for DC-precursors such as monocytes and immature DC:s. It has moreover been shown that a local injection of a plasmide vaccine expressing GM-CSF induces a local accumulation of immature DCs at the vaccination site that is followed by the appearance of mature DCs in regional lymph nodes, consistent with egression of maturating DC from the injection site and migration to the draining lymph nodes. The indirect pathway of tumor antigen presentation also solves the problem with the demand for CD4+ T helper type-1 (Th1) cells in order to achieve an efficient and long-lasting tumor-specific CTL immune response since also MHC class II restricted CD4+ cells can be activated via the indirect pathway.
With this knowledge at hand it was now open for refining this principle, ex vivo and in vivo, which is based upon an efficient indirect presentation of tumor antigen by host (autologous) APC. An obvious approach has been to propagate potent APCs ex vivo, in particular dendritic cells (DCs), and thereupon load these cells with tumor-derived proteins either by pulsing or transfection. Also so called dendritoma vaccines, where autologous tumor cells have been fused with autologous monocyte-derived DCs, have been developed. A problem with ex vivo propagation of autologous DCs is however the minor migration of these cells to draining lymph nodes (a prerequisite for the injected DCs to meet naive T cells). Studies in humans have shown that 1% maximum of subcutanously injected DCs migrates to regional lymph nodes. An alternative approach has therefore been developed, above all from the observations during vaccination with GM-CSF producing tumor cells in rodent models, with the goal to induce an efficient recruitment in vivo of host DCs to the vaccination site. Phase I trials in humans with prostate and renal carcinoma and melanoma using autologous GM-CSF transfected tumor cells vaccines have been evaluated and found to be safe but without any obvious clinical effect. Additionally, a vaccine based upon autologous tumor cells have been shown to create several technical problems. Firstly, the vaccine depends on the availability of adequate numbers of tumor cells, which are rarely available because of the reactive process that are found infiltrating tumor cells of many common cancers. Secondly, the vaccine requires de novo gene transfer for treatment of each patient, which is labor intensive and may cause variable cytokine expression levels between different patient vaccines. Thirdly, there is significant expense and time required to certify each patient's lot of vaccine so that they meet accepted administration guidelines. One way to circumvent these technical obstacles is to use a vaccine strategy that is based on a panel of cytokine-expressing allogeneic tumor cell lines that can be formulated and stored before the initiation of clinical studies. This is a particularly attractive approach for the majority of common cancers for which specific tumor antigens have not yet been identified. Two findings provide the immunologic rationale for an allogeneic tumor-cell vaccine approach. Firstly, that DCs of the host, rather than the vaccinating tumor themselves, are responsible for priming of CD4+ cells and CD8+ CTL, both of which are required for generating systemic antitumor immunity (see above). Secondly, many tumor antigens are commonly expressed among different patient's tumors. A GM-CSF vaccine based on this concept has been developed in rodent models (Chang E. Y. et al, (International Journal of Cancer, 2000, Vol 86. No. 5, pp 725-730) and was recently studied in a Phase I trial and found to be safe but without any obvious clinical effect. Most likely, this clinical insufficiency was due to the production of one single cytokine (GM-CSF) since theoretically several factors ought to be produced locally in order to induce not only an efficient recruitment of immature DCs but also an efficient maturation of these cells. Necessary factors most probably include chemotactic cytokines such as MIP-1 alpha and/or RANTES, maturation factors such as interleukin (IL)-1 beta, IL-6 and/or tumor necrosis factor (TNF) alpha and finally Th1-polarizating factors such as interferon (IFN) gamma.
Within the transplantation and transfusion areas there is daily struggling with the problem of allo-immunization, which is a T-cell-mediated immunization against indirectly presented donor-specific HLA-antigens. Such immunization is frequently developed with a strong power after transfusions with allogeneic blood products and after transplantation of solid organs. Not even a powerful continuous immunosuppressive treatment after a primary successful transplantation of a HLA-incompatible organ appears to prevent a slowly progressing process referred to as chronic rejection. This process is mediated by CD4+ cells of Th1-type, which are activated by allogeneic HLA-peptides indirectly presented by autologous APCs. These CD4+ T cells in turn activate donor-specific antibody (IgG)-producing B-cells and cytotoxic T cells and tissue macrophages, which constitute the different effector mechanisms during chronic rejection. A very central actor for the starting-up of an allo-immunization appears to be viable, metabolically intact, donor-derived (allogeneic) APCs. If these are depleted or inactivated by UVB-irradiation before a transfusion of e.g. platelet concentrates then the immunization is usually avoided. This also pertain passenger APCs in transplanted tissue; if these allogeneic APCs are depleted before transplantation the risk for a subsequent immunization with chronic rejections is essentially decreased. For non-viable allogeneic tissue the same immunization rules pertains as for other foreign protein-derived antigens i.e. in order to achieve an essential immunization it is necessary to administer the antigen in a relatively large amount together with an adjuvant as e.g. Freuds complete adjuvant (FCA). The same is valid for in vitro primary stimulation with non-viable MHC-expressing allogeneic APCs or pure MHC-derived allo-peptides which do not induce any substantial priming of naive T cells indirectly recognizing allo-derived peptides. A substantial priming of these T cells is however obtained by using viable allogeneic MHC-expressing APC during the primary stimulation.
Something that differ (discriminates) primary stimulation with viable allogeneic APCs from primary stimulation with non-viable (lysed or apoptotic) allogeneic APCs in vitro is the very powerful T cell proliferation in the responder (host) cell population, only seen during stimulation with viable APCs. This reaction which is called allogeneic mixed leukocyte reaction (MLR) also leads to the production of certain chemokines and cytokines, including MIP-1 alpha, RANTES, IL-1beta, IL-6, TNF-alpha and IFN-gamma. The MLR is induced in both naive and memory responder T-cells that are cross-reacting with MHC molecules on allogeneic APCs through experiencing these molecules as their own MHC+ foreign peptide sequences which the T-cells that were predestined to react against. It has been shown that as many as 1 out of 20 of our circulating T cells may participate in this preformed allo-reactivity. It is further known that treatment of stimulator-APC with agents that reduce or remove sialic acids from glycoproteins on the cell membrane, such as neuraminidase and anti-CD43 antibodies, increase the potential of APC to induce a proliferative response in allogeneic T cells.
A method for inducing an antigen-specific immune response by co-administration of allogeneic DCs with autologous DC, in which the antigen was incorporated, to a subject is disclosed in WO 99/47687. The autologous DC were expected to present the antigen to CTLs while the allogeneic DC were expected to induce a strong reaction from alloreactive T cells resulting in the local release of stimulatory molecules that would amplify the ability of autologous DC to activate CTLs. No data supporting their hypothesis was presented nor any immune response elicited.
Vaccination with hybrid cells consisting of autologous tumor cells fused with allogeneic mature DC is described in “Regression of human metastatic cell carcinoma after vaccination with tumor cell-dendritic cell hybrids”, A. Kugler et al. Nature Medicine, vol 6, No 3, March 2000, pp. 332-336. Theoretically, this method is based upon the expectance that co-expression of allogeneic MHC molecule on the semi-allogeneic tumor cell (expressing autologous MHC class I molecules+tumor peptides) would activate alloreactive T cells. This activation would result in a local release of stimulatory cytokines that would help to trigger the activation of CTLs recognizing the tumor peptide on autologous, tumor cell-derived, MHC class I molecules. Using this vaccine approach a limited number of patients with renal tumor exhibited a clinical anti-cancer response.
In WO 9421798 it is mentioned that transfection of DNA encoding neuraminidase protein into autologous APCs (but not allogeneic APCs) could be used to boost their ability to present tumor antigens directly to autologous, MHC-restricted, T cells (see page 3 line 20-23 and line 28, page 4 line 2 and page 7 line 22-26). This concept is based upon the central dogma within immunology put forward by Zinkernagl and Doherty in the middle of the 70's: A T-cell only recognize foreign peptides (e.g. tumor-derived peptides from an autologous or allogeneic tumor) if they are presented by APCs which express own MHC molecules (i.e MHC-compatible APC), so called “self MHC-restriction”.
Methods utilizing the immunogenicity of allogneic APCs as adjuvant when vaccinating with antigen-loaded autologous APCs have earlier been disclosed as said above. In WO 99/47687, autologous antigen-loaded APC were expected to present the antigen to MHC-restricted autologous CTLs while the co-administered allogeneic APC were expected to induce an inflammatory allogeneic response that would amplify the ability of aotologous DC to activate CTLS.
A vaccine against other tumors, using dendritic cells fused with cancer cells, is also suggested in “Smallpox, polio and now a cancer vaccine?”, D. W. Kufe, Nature Medicine, vol 6, No 3, March 2000, pp. 252-253. The methods of Kugler et al and Kufe above are however limited by a number of factors. Firstly, an autologous vaccine depends on the availability of adequate numbers of tumor cells, which are rarely available because of the reactive process that is found infiltrating tumor cells of many common cancers. Secondly, an autologous vaccine requires de novo gene transfer for treatment of each patient, which is labor intensive and may cause variable cytokine expression levels between different patient vaccines. Thirdly, there is significant expense and time required to certify each patient's lot of vaccine so that they meet accepted administration guidelines.
In US 20020039583 A1 there is further mentioned allogeneic and also xenogeneic APCs loaded with immune complex containing stress proteins as a thinkable cellular vaccine.
The methods disclosed in WO 99/47687 and in Nature Medicine by Kugler at al are however limited by a number of factors. First both methods are dependent on the availability of adequate numbers of autologous cells (APCs and/or tumor cells). Secondly, both methods require labour-intensive manipulations of autologous cells for treatment of each patient. Thirdly there is significant expense and time required to certify each patent's lot of vaccine so that they meet accepted administration guidelines.
Accordingly there is a need for a vaccine that creates a better immune.
response and is better suited for storing, producing in a large scale and is independent of supply limitations.